JP2011084516A - Glycosaminoglycan decomposition promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein having human-derived glycosaminoglycan decomposition activity with no fear of mixing of a protease and the like as a drug or the like instead of chondroitinase produced by microorganisms; and a method for producing a functional oligosaccharide using this protein. <P>SOLUTION: The glycosaminoglycan decomposition promotor includes a protein comprising an amino acid sequence shown in sequence ID No.1 or a protein comprising an amino acid sequence shown in sequence ID No.1 in which one or several tens of amino acids are substituted, deleted or added, and having activity of hydrolyzing the N-acetyl-D-galactosaminide bonding of sulfated N-acetyl-D-galactosamine bonded to D-glucuronic acid as an active ingredient. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a glycosaminoglycan degradation promoter containing a protein derived from human-derived glycosaminoglycan hydrolysis activity as an active ingredient, and a pharmaceutical use thereof.

  Chondroitinase and hyaluronidase are known as enzyme proteins that degrade glycosaminoglycans (mucopolysaccharides), which are long-chain polysaccharides having amino sugars such as chondroitin and hyaluronan. Among them, chondroitinase catalyzes a reaction for decomposing glycosaminoglycans such as hyaluronan, chondroitin sulfate, chondroitin, and dermatan sulfate into disaccharides containing unsaturated sugars.

  So far, chondroitinase derived from various organisms has been reported. Representative examples include chondroitinase ABC derived from Proteus vulgaris (CaseABC; Patent Document 1), chondroitinase AC derived from Flavobacterium heparinum (CaseAC; non-patent document 1), chondroitinase ACII derived from Arthrobacter aurescens ACII Non-Patent Document 2), Flavobacterium sp. Examples include chondroitinase ACIII derived from Hp102, chondroitinase C (CSase ACIII, CSaseC; non-patent document 3), chondroitinase B derived from Flavobacterium heparinum (CSaseB; non-patent document 4), and the like.

  CSaseABC (EC 4.2.2.4) degrades not only chondroitin sulfate A derived from mammalian cartilage, chondroitin sulfate C derived from shark cartilage and chondroitin sulfate B derived from mammalian skin (dermatan sulfate), but also hyaluronan. And is commercially available as a reagent for removing glycosaminoglycans from animal tissues and a reagent for identifying glycosaminoglycans in tissues.

  Recently, an attempt has been made to use chondroitinase, in particular CSaseABC or CSaseAC, as a therapeutic agent for intervertebral hernia that is directly administered to the intervertebral disc space (Patent Document 2). Conventionally, intervertebral discolytic therapy (ID therapy) has been used in which plant papaya-derived proteolytic enzyme chymopapain, bacteria-derived collolytic enzyme collagenase, and the like are injected into the disc space of hernia patients. However, it is pointed out that ID therapy using proteolytic enzymes suffers from side effects such as nerve palsy and allergy due to the degradation of the protein part of the surrounding structural tissues as well as the hernia part of the spine and intervertebral disc. Has been.

  Therefore, there are many expectations for spinal injury treatment using chondroitinase having glycosaminoglycan degrading enzyme activity instead of proteolytic enzyme. However, most of the known chondroitinases are obtained from microorganisms that simultaneously produce proteolytic enzymes. CaseABC is a typical example. The microbial culture contains protease activity, endotoxin activity, and nucleic acids, so the use of microorganism-derived chondroitinase as a high-purity reagent for administration to the human body as a therapeutic agent for disc herniation Has inconvenient aspects.

  On the other hand, the present inventors have disclosed Ce-Chase which is an enzyme protein having nematode-derived chondroitinase activity (Non-patent Document 5). However, in the case of an enzyme protein having human-derived chondroitinase activity, Absent.

  Furthermore, it has been suggested that the human HYAL4 (Hyaluronoglucosaminedase 4) gene, which has been a gene encoding an enzyme protein having hyaluronidase activity, may encode a chondroitin sulfate-specific enzyme protein (Non-patent Document 6). There is no specific disclosure of what kind of enzyme it is.

  In addition, glycosaminoglycans, which are long-chain polysaccharides, have been pointed out for their usefulness in moisture retention, lubricity and other properties, and various health foods and pharmaceuticals containing glycosaminoglycans have been developed. Oligosaccharides obtained by lowering the molecular weight instead of sugars are expected to have various physiologically active functions.

JP-A-6-153947 U.S. Pat. No. 4,696,816

Nakagawa Yuri et al., Obihiro Institute I, 17, 7-12 (1990) Hiyama et al. Biol. Chem. 1975, 250, 1824 Hirofumi Miyazono et al., “Biochemistry”, 1989, 61, 1023 Michelacci et al., Biochem. Biophys. Res. Commun. 1974, volume 56, page 973. Kaneiwa et al. Biol. Chem. 2008, 283, 14971 Csoka et al., Matrix Biol. 2001, Vol. 20, p. 499

  In the present invention, instead of chondroitinase produced by microorganisms, a human-derived protein having glycosaminoglycan degrading activity with a low risk of contamination such as proteolytic enzyme is provided as a pharmaceutical, etc. It aims at providing the manufacturing method of the used functional oligosaccharide.

  In the course of research on the physiological function of chondroitin in humans, the present inventors have found that an enzyme protein having chondroitinase activity exists in humans, and have completed the following inventions.

(1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or consisting of an amino acid sequence in which one or several tens of amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, A glycosaminoglycan degradation accelerator comprising a protein having an activity of hydrolyzing an N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine bound to D-glucuronic acid as an active ingredient.

(2) a protein comprising an amino acid sequence in which amino acid residues 1 to 33 and / or 463 to 481 in SEQ ID NO: 1 are deleted, or amino acids 1 to 33 and / or 463 to 481 in SEQ ID NO: 1 Glycosaminoglycan degradation promotion according to (1), wherein the active ingredient is a protein comprising an amino acid sequence in which a residue is deleted and one or several amino acids are substituted, deleted, or added Agent.

(3) The glycosaminoglycan degradation accelerator according to (1) or (2), wherein the glycosaminoglycan is chondroitin sulfate or a hybrid polysaccharide containing chondroitin sulfate.

(4) The glycosaminoglycan degradation according to (3), wherein the chondroitin sulfate is one or more chondroitin sulfates selected from the group consisting of chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D and chondroitin sulfate E Accelerator.

(5) The glycosaminoglycan degradation accelerator according to any one of (1) to (4), wherein the protein is in the form of a fusion protein.

(6) A pharmaceutical composition comprising the glycosaminoglycan degradation accelerator according to any one of (1) to (5) and a pharmaceutically inactive ingredient.

(7) The pharmaceutical composition according to (6), for treating spinal cord injury.

(8) The pharmaceutical composition according to (7), wherein the spinal injury is an injury caused by a herniated disc.

(9) The pharmaceutical composition according to (8), wherein the intervertebral disc herniation is epidural migration type intervertebral disc herniation or transligament prolapse disc herniation.

(10) A method for producing an oligosaccharide having glucuronic acid as a non-reducing end from glycosaminoglycan, wherein the protein consists of the amino acid sequence shown in SEQ ID NO: 1, one in the amino acid sequence shown in SEQ ID NO: 1 or The N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine consisting of an amino acid sequence in which several tens of amino acids are substituted, deleted, or added and linked to D-glucuronic acid is hydrolyzed. A protein having an activity of degrading, a protein comprising an amino acid sequence in which amino acid residues 1 to 33 and / or 463 to 481 are deleted in SEQ ID NO: 1, and 1 to 33 and SEQ ID NO: 1 to 463 to 463 The 481st amino acid residue was deleted, and one or more amino acids The activity of hydrolyzing the N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine consisting of an amino acid sequence in which an acid is substituted, deleted or added and bound to D-glucuronic acid. The method comprising the steps of reacting one or more proteins selected from the group consisting of proteins having a glycosaminoglycan and recovering an oligosaccharide having glucuronic acid as a non-reducing end.

(11) The method for producing an oligosaccharide according to (10), wherein the glycosaminoglycan is chondroitin sulfate or a hybrid polysaccharide containing chondroitin sulfate.

(12) The oligosaccharide according to claim 11, wherein the chondroitin sulfate is one or more chondroitin sulfates selected from the group consisting of chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D and chondroitin sulfate E. Method.

  The glycosaminoglycan degradation promoter of the present invention can be used as a glycosaminoglycan degradation promoter containing a human-derived enzyme protein that does not contain bacterial toxins and the like as an active ingredient for the treatment of spinal disorders and the like. In addition, oligosaccharides produced using proteins that are glycosaminoglycan degradation accelerators are oligosaccharides having various structures having glucuronic acid at the non-reducing end, and are antigenic to mammals, particularly humans. Low functional oligosaccharides can be produced.

It is the figure which performed the silver dyeing | staining method and the western method about the protein expressed by the recombinant cell. Lanes 1 to 3 show the results of silver staining. Lanes 4 and 5 show the results of the Western method under reducing conditions, and lanes 6 and 7 show the results of the Western method under non-reducing conditions. Lanes 2, 4, and 6 are the results obtained with the protein obtained from the culture medium of the recombinant cells, and lanes 3, 5, and 7 are the results obtained with the protein obtained from the culture medium of the cells not subjected to recombination as controls. . Lane 1 is a molecular weight marker. FITC-labeled hyaluronic acid (Panel A), CS-A (Panel C), CS-C (Panel E), CS-D (Panel G) gel filtration elution patterns before enzymatic reaction, and enzyme reaction It is a figure which shows the elution pattern (panel B, D, F, and H) of the gel filtration of the decomposition product of (2). V ₀ indicates a void, and V _t indicates a total capacity. It is a figure which shows the elution pattern of the gel filtration of the decomposition product fluorescently labeled by 2AB. Panel A shows the results of CS-D, Panel B shows the results of CS-C, and Panel C shows the results of CS-A. V ₀ represents a void, and V _t represents a total capacity. In addition, each peak indicated by ▼ in the figure is fractionated to be extracts D-1, D-2, and D-3, respectively. It is a graph which shows the optimal pH of CS-D degradation activity of human CSHY which is a protein which has glycosaminoglycan degradation activity. It is a graph which shows the Lineweaver-Burk plot created using the initial reaction rate at the time of hydrolyzing CS-A, CS-C, CS-D, and CS-E with human CSHY. . Graphs A, B, C and D show the results of CS-A, CS-C, CS-D and CS-E, respectively. Extracts D-1, D-2 and D-3, which are human CSHY degradation products of CS-D, are digested with CSase AC-II, and the resulting degradation products are fluorescently labeled with 2AB and anion exchange chromatography is performed. It is a figure which shows the result of having analyzed. It is a figure which shows the result of having digested the human CSHY degradation product of CS-A and CS-C with CSase AC-II, and fluorescent-labeling the obtained degradation product with 2AB, and analyzing it by anion exchange chromatography. It is a schematic diagram which shows the structure of the site | part cut | disconnected by human CSHY in CS-C. It is a figure which shows the decomposition | disassembly activity of mutant human CSHY with respect to CS-D. Panels A, B, C, D, E and F show the results when using inactive control, mutant 1, mutant 2, mutant 4, mutant 3, and active control (wild type), respectively. .

  Hereinafter, the glycosaminoglycan degradation accelerator according to the present invention will be described in detail. The glycosaminoglycan degradation accelerator of the present invention comprises a human-derived protein consisting of the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient.

The human HYAL4 (Hyaluronoglucosaminoidase4) gene encoding the amino acid sequence of SEQ ID NO: 1 was registered in 2008 as GenBank ^™ accession number NM — 012269. This human HYAL4 gene has a 638 bp untranslated region at the 5 ′ end, a 1446 bp ORF corresponding to 481 amino acid residues having four N-glycosylation sites, and a 327 bp untranslated region at the 3 ′ end. have. In addition, using a SOSUI System (http://bp.nuap.nagoya-u.ac.jp/sosui/) for predicting the secondary structure of a membrane protein, the second protein of the amino acid sequence encoded by the ORF is used. From the analysis of the following structure, the protein is presumed to be a type I transmembrane protein having a hydrophobic region consisting of 33 amino acid residues at the N-terminus and a GPI anchor region consisting of 19 amino acid residues at the C-terminus. The

Furthermore, by searching for a protein homologous to the amino acid sequence encoded by the ORF with respect to a database relating to the amino acid sequence information of the protein, human-derived hyaluronidase (GenBank ^™ accession number NM_007312) has a 40% homology. Identified as a protein having However, the function or physiological activity of the protein consisting of the amino acid sequence of SEQ ID NO: 1 remains unknown.

  In addition, as described above, Csoka et al. Suggested that the HYAL4 gene encodes an enzyme protein peculiar to chondroitin sulfate. However, what kind of enzyme is disclosed specifically. (Non-patent document 6).

  As a result of subsequent studies, the present inventors have found that the protein comprising the amino acid sequence of SEQ ID NO: 1 hydrolyzes the N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine bound to D-glucuronic acid. The protein expressed using recombinant cells to have a degrading activity and to exhibit glycosaminoglycan, particularly a glycosaminoglycan composed of a mixed chain containing chondroitin sulfate or chondroitin sulfate. This was confirmed through experiments using proteins. The present invention has been completed based on this finding. Hereinafter, the protein consisting of the amino acid sequence of SEQ ID NO: 1 is represented as human CSHY.

  The identity between the amino acid sequence of the known chondroitinase and the amino acid sequence of SEQ ID NO: 1 is as follows. When the amino acid sequence of SEQ ID NO: 1 is 100, CSaseABC derived from Proteus vulgaris is 0.5%, CaseAC derived from Flavobacterium heparinum Is 5.7%, Arthrobacter aurescens-derived CSase ACII is 2.5%, and Flavobacterium heparinum-derived CSase B is 5.3%. Thus, the human CSHY of the present invention has a characteristic amino acid sequence that shows little identity to known chondroitinases.

  The glycosaminoglycan degrading activity of human CSHY can be detected by gel filtration or other methods of degradation products produced by reacting human CSHY in a suitable buffer with a glycosaminoglycan such as chondroitin sulfate as a substrate. Can be confirmed. In particular, the substrate glycosaminoglycan is labeled in advance using FITC (fluorescein 5 (6) isothiocyanate) or other appropriate labeling reagent, and the decomposition product is detected using fluorescence or other signals. By quantifying, the degradation activity of glycosaminoglycan can be easily measured and further quantified.

  A product obtained by reacting human CSHY expressed as a recombinant protein using COS7 cells with various glycosaminoglycans labeled with FITC, or reacting human CSHY with various glycosaminoglycans The enzymological properties of human CSHY confirmed by labeling with 2-aminobenzamide (2AB) are as follows.

1) Human CSHY has an activity to hydrolyze the N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine bound to D-glucuronic acid. As a result, the glycosaminoglycan having a mixed chain containing chondroitin sulfate or chondroitin sulfate is degraded. In particular, it exhibits high degradation activity against chondroitin sulfate and hardly exhibits degradation activity against chondroitin. The level of degradation activity is chondroitin sulfate D> chondroitin sulfate C> chondroitin sulfate A> chondroitin sulfate E. Assuming that the affinity for human CSHY when chondroitin sulfate D is used as a substrate is 100, the affinity when chondroitin sulfate C, chondroitin sulfate A or chondroitin sulfate E is used as a substrate is 50, 33.3 and 5, respectively. is there. For glycosaminoglycans having a hybrid chain containing chondroitin sulfate, the degradation activity varies depending on the mixture ratio of chondroitin sulfate and other glycosaminoglycans.

2) For example, the product of the degradation reaction of chondroitin sulfate D by human CSHY is GlcAβ1-3GalNAc (4S) β1-4GlcA (2S) β1-3GalNAc (6S) (“GlcA” is D-glucuronic acid, “GalNAc” Is N-acetyl-D-galactosamine, “β1-3” is β1-3 bond, “β1-4” is β1-4 bond, “GalNAc (4S) or GalNAc (6S)” is position 4 of GalNAc Or “GlcA (2S)” indicates that the 2-position of GlcA is sulfated, and the reducing end is GalNAc (N-acetyl-D-galactosamine). is there. This result indicates that human CSHY is a kind of endo-β-galactosaminidase that is a hydrolase. In addition, all known chondroitinases are derived from bacteria, and are not hydrolases but a detachment enzyme (eliminase). The fact that there is little identity between the known bacterial chondroitinase and the human CSHY of the present invention is presumed to be related to the difference in the reaction mechanism.

3) The preferable pH of chondroitin sulfate degradation activity of human CSHY is pH 4.5 to 6.5, more preferable pH is pH 4.5 to 6.0, and further preferable pH is pH 4.5 to 5.5. A preferred pH is 5.0.

  Thus, compared with conventional CSaseABC, BC, ACII, etc., which is a kind of desorption enzyme that strongly degrades chondroitin sulfate A, chondroitin sulfate C and dermatan sulfate and weakly degrades hyaluronan, One human CSHY is an enzyme protein that hydrolyzes the N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine bound to D-glucuronic acid, and has a high degrading activity especially on chondroitin sulfate. .

  In addition to the above-described human CSHY, that is, a glycosaminoglycan degradation accelerator containing the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient, the present invention has one amino acid sequence shown in SEQ ID NO: 1. Alternatively, the N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine consisting of an amino acid sequence in which several amino acids are substituted, deleted, or added and linked to D-glucuronic acid is hydrolyzed. Disclosed is a glycosaminoglycan degradation accelerator comprising a protein having an activity of degrading as an active ingredient.

  The term “one or several tens” relating to substitution, deletion, and / or addition of an amino acid sequence in the present invention is within 1 to several tens of amino acids, preferably 1 to 70, more preferably 1 to 50, still more preferably. Means a change of 1 to 30, particularly preferably 1 to 15 amino acid residues. Expressed in terms of amino acid sequence identity (%), the amino acid sequence represented by SEQ ID NO: 1 has 80% or more, preferably 85% or more, more preferably 90% or more, and particularly preferably 95% or more identity. It can be expressed as an amino acid sequence having.

  It has been empirically recognized that the amino acid sequence of a protein can tolerate highly conservative variations in physicochemical properties such as the charge, size, and hydrophobicity of amino acid residues. For example, for substitution of amino acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln) ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg). It is also experienced by those skilled in the art that even when the above-described conservation is exceeded, mutations that do not lose the essential function of the protein can exist.

  Therefore, in the amino acid sequence shown in SEQ ID NO: 1, even a protein comprising an amino acid sequence in which one or several tens of amino acids are substituted, deleted, and / or added, sulfation bound to D-glucuronic acid In some cases, N-acetyl-D-galactosamine has an activity of degrading the activity of hydrolyzing the N-acetyl-D-galactosaminide bond, and the use of such a protein as a glycosaminoglycan degradation accelerator is the present invention. It is one aspect | mode. For example, as will be described in detail later, human CSHY comprising an amino acid sequence from which the hydrophobic region comprising the N-terminal 33 amino acid residues and / or the C-terminal 19 amino acid residues of the amino acid sequence represented by SEQ ID NO: 1 has been deleted Retains the activity of degrading chondroitin sulfate. Such a protein from which the N-terminal 33 amino acid residues and / or the C-terminal 19 amino acid residues have been deleted is also an embodiment of human CSHY according to the present invention.

  That is, the present invention relates to a protein comprising an amino acid sequence from which amino acid residues 1 to 33 and / or 463 to 481 are deleted in SEQ ID NO: 1, or from 1 to 33 and / or 463 to 481 in SEQ ID NO: 1. A sulfated N-acetyl-D- consisting of an amino acid sequence with one or several amino acid residues deleted, further substituted, deleted or added, and linked to D-glucuronic acid The present invention also provides a protein having an activity of hydrolyzing the N-acetyl-D-galactosaminide bond of galactosamine, and further a glycosaminoglycan degradation accelerator containing these proteins as an active ingredient.

  Moreover, said human CSHY can be manufactured as what is called a fusion protein which added other protein or polypeptide to the N terminal and / or C terminal, and can be utilized as a glycosaminoglycan degradation promoter. Such a fusion protein and its use as a glycosaminoglycan degradation accelerator are also an embodiment of the present invention. Such a fusion protein has increased utility as compared to the case where human CSHY is produced or used alone, in that the function exhibited by other proteins or polypeptides is added to human CSHY.

Examples of proteins include glutathione S transferase (GST), maltose binding protein (MBP), protein A, green fluorescent protein (GFP), luciferase, preprotrypsin, and other proteins commonly used in the production of fusion proteins. it can. In addition, if a polypeptide that facilitates the production of a recombinant protein, in particular, the purification of the recombinant protein, such as a FLAG ^(R) tag, a histidine tag or a chitin-binding sequence, is utilized, human CSHY can be produced more advantageously. it can.

  Furthermore, it is possible to add an appropriate labeling compound such as a fluorescent substance or a radioactive substance to human CSHY, or to bind various chemically modified substances or polymers such as polyethylene glycol to the human CSHY. Alternatively, human CSHY can be bound to an insoluble carrier. Chemical modification methods for such proteins are widely known to those skilled in the art, and may be modified and used in any way as long as the function of the protein used in the present invention is not impaired. A glycosaminoglycan degradation accelerator containing human CSHY having such a modification as an active ingredient is also an aspect of the present invention.

  The human CSHY according to the present invention may be directly purified from human cells and used, but can be produced as a recombinant protein using a nucleic acid encoding human CSHY and a DNA consisting of the base sequence shown in SEQ ID NO: 2. preferable.

  The nucleic acid consisting of the base sequence shown in SEQ ID NO: 2 is designed using the human genomic DNA library or cDNA library as a template, and designed and synthesized appropriate PCR primers based on the base sequence information shown in SEQ ID NO: 2. Can be cloned by performing a PCR reaction according to a known method (see, for example, Michael AI, et al., PCR Protocols, a Guide to Methods and Applications, Academic Press, 1990). A nucleic acid having the base sequence shown in SEQ ID NO: 2 is produced by a chemical synthesis method such as a phosphoramidite method or a commercially available DNA synthesizer based on the base sequence information disclosed in this specification. You can also

  Human CSHY is not particularly limited when it is used after being directly purified from human cells, or when a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 2 is cloned using a DNA library or cDNA library as a template. However, in this example, the cloning is performed using a human placenta cDNA library as a template.

  The DNA encoding human CSHY according to the present invention can be recombined into an appropriate expression vector, and such a recombinant vector is used for the recombinant production of the protein used in the present invention. The recombinant vector may be in any form such as circular or linear. Such a recombinant vector may have other nucleotide sequences if necessary in addition to the nucleic acid encoding the protein used in the present invention. Other base sequences include leader sequences, enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used for the purpose of copy number amplification, base sequences encoding signal peptides, base sequences encoding other polypeptides A poly A addition sequence, a splicing sequence, a replication origin, a base sequence of a gene serving as a selection marker, and the like.

  Methods for gene recombination performed when creating a fusion protein or mutant protein include adding a translation initiation codon and translation termination codon to the nucleic acid encoding human CSHY using an appropriate synthetic DNA adapter, or bases. It is also possible to newly generate or eliminate a suitable restriction enzyme cleavage sequence within the sequence. These are within the scope of operations normally performed by those skilled in the art, and those skilled in the art can arbitrarily and easily process them based on DNA encoding human CSHY.

  In addition, the vector holding the nucleic acid encoding the protein used in the present invention may be selected from an appropriate vector according to the host to be used. In addition to the plasmid, bacteriophage, baculovirus, retrovirus, vaccinia It is also possible to use various viruses such as viruses.

Available commercial expression vectors include pcDM8 (Funakoshi), pcDNAI (Funakoshi), pcDNAI / AmP (Invitrogen), EGFP-C1 (Clontech), pREP4 (Invitrogen), pGBT-9 (Clontech) PGEM ^(R) -T Easy (Promega), p3XFLAG-CMV-8 (Sigma), and the like.

  Expression of the protein used in the present invention can be expressed under the control of a promoter sequence specific to the gene encoding the protein. Alternatively, another appropriate expression promoter can be linked upstream of the base sequence encoding the protein used in the present invention.

  The expression promoter may be appropriately selected according to the host and the purpose of expression. For example, when the host is Escherichia coli, T7 promoter, lac promoter, trp promoter, λPL promoter, etc., and when the host is yeast, PHO5 When the host is an animal cell, the promoter, GAP promoter, ADH promoter, etc. are SV40-derived promoter, retrovirus promoter, cytomegalovirus (human CMV) IE (immediate early) gene promoter, metallothionein promoter, heat shock promoter And SRα promoter.

  An operation such as linking a nucleic acid encoding a protein used in the present invention, preferably DNA, to the promoter exemplified above or incorporating it into an expression vector is described in J. Org. Sambrook et al. {Molecular Cloning, a Laboratory Manual 2nd ed. , Cold Spring Harbor Laboratory, New York, 1989}, and the like, can be performed based on the instructions in the experimental manual describing in detail various gene recombination operations.

  Examples of host cells include Escherichia, Corynebacterium, Brevibacterium, Bacillus, Serratia, Pseudomonas, Pseudomonas Genus bacteria, Arthrobacter genus, Erwinia genus bacteria, Methylobacterium genus bacteria, Rhodobacter genus bacteria, Streptomyces genus microorganisms, Zymomonas Microorganisms such as yeasts of the genus Saccharomyces, Insect cells such as ko, HEK293 cells, MEF cells, Vero cells, Hela cells, CHO cells, WI38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, HKG cells, human kidney cell lines, etc. Can be mentioned. Among them, the use of eukaryotic cells, particularly mammalian cells such as COS-7 cells is preferred.

As a method for introducing an expression vector into a host cell, methods described in the above-mentioned experimental manuals such as Sambrook et al., For example, electroporation method, protoplast method, alkali metal method, calcium phosphate precipitation method, DEAE In addition to the dextran method, microinjection method, particle gun method, etc., for example, Nupherin ^™ -Neuron (BIOMOL), HiyMax (Dojin Chemical Laboratories), GeneJuice ^™ Transfection Reagent (Novagen), jetPEI (Polyplasts) company), PolyMag (OZ Biosciences, ^Inc.), Lipofectamine TM LTX (Invitrogen ^), TransFast TM Transfection Reagent (Promega ^{Corp.), FuGENE (R) HD Transfection} Reagent (Roche diagnostics , ^Inc.), can be performed using commercially available transfection reagents such as FuGENE (R) 6 (Roche diagnostics, Inc.). For the use of insect cells such as Sf9 and Sf21, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York, 1992). And Bio / Technology, 1988, Volume 6, Page 47, and the like.

  The protein used in the present invention can be obtained by expressing the above expression vector in the above host cell, collecting the target protein from the host cell or culture medium, and purifying it. As a method for purifying a protein, an appropriate method can be appropriately selected from methods usually used for protein purification. That is, various affinity chromatography such as salting-out method, ultrafiltration method, isoelectric precipitation method, gel filtration method, electrophoresis method, ion (anion, cation) exchange chromatography, hydrophobic chromatography and antibody chromatography, A suitable method is appropriately selected from methods that can be generally used such as chromatofocusing method, adsorption chromatography, and reverse phase chromatography, and if necessary, using a high performance liquid chromatography (HPLC) system or the like. Purification may be performed in an appropriate order.

  When the protein used in the present invention is expressed as a fusion protein with another functional protein or polypeptide, it is preferable to employ a purification method characteristic of the functional protein or polypeptide. The fusion protein can be cleaved with an appropriate protease (thrombin, trypsin, etc.) to recover the protein of the present invention.

  As described above, the protein used in the present invention can be prepared in the form of a single protein alone or in the form of a fusion protein with another type of protein, but is not limited thereto, and is used in the present invention. It is also possible to convert the protein into various forms. For example, various chemical modifications to proteins, binding to a polymer such as polyethylene glycol, binding to an insoluble carrier, and encapsulation in liposomes can be considered by various techniques known to those skilled in the art.

  The protein used in the present invention is an organic chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBoc method (t-butyloxycarbonyl method), or a suitable commercially available peptide. It can also be produced using a synthesizer.

  The present invention provides a glycosaminoglycan degradation accelerator containing human CSHY as an active ingredient, and a pharmaceutical composition comprising the glycosaminoglycan degradation accelerator and a pharmaceutically inactive ingredient. The pharmaceutical composition is a pharmaceutical composition for treating spinal cord injury, particularly spinal injury caused by intervertebral disc herniation, more preferably epidural migrating disc herniation or transligament prolapse disc herniation.

  The content of human CSHY in the pharmaceutical composition may be an effective amount for decomposing or dissolving a substance containing glycosaminoglycan that damages the spine, specifically, nucleus pulposus. Here, the “effective amount” means an amount capable of dissolving the nucleus pulposus existing in the spinal epidural space by protrusion, migration, etc., and eliminating the influence of the nucleus pulposus.

  Pharmaceutically inactive ingredients are not themselves active ingredients for therapeutic purposes, i.e., have no pharmacological activity, but are carriers, excipients, binders, lubricants, colorants, disintegrants, buffering agents. Ingredients commonly used in the preparation of drugs or pharmaceutical compositions, such as isotonic agents, preservatives and the like.

  For example, carriers or excipients include dextrans, sucrose, lactose, maltose, xylose, trehalose, mannitol, xylitol, sorbitol, inositol, serum albumin, gelatin, polyethylene glycol, nonionic surfactants (for example, polyoxy Ethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, sucrose fatty acid ester, polyoxyethylene polyoxypropylene glycol) and the like.

  The blending ratio of human CSHY and the pharmaceutically inactive ingredient in the composition of the present invention is not particularly limited, and can be appropriately determined by those skilled in the art according to the dose, the form of the composition, and the like. Further, the glycosaminoglycan degradation accelerator of the present invention or a pharmaceutical composition containing the same may be in the form of a solution, a solid agent, a paste, or other forms, and they adopt a pharmacologically known method. Thus, it can be appropriately prepared. A preferred form is a form of an injectable preparation containing human CSHY, particularly a lyophilized preparation. Such a preparation can be produced by a general method for preparing an injectable preparation containing a protein, particularly a lyophilized preparation.

  The glycosaminoglycan degradation accelerator of the present invention and a pharmaceutical composition containing the glycosaminoglycan degradation accelerator and a pharmaceutically inactive component include sulfated N bound to D-glucuronic acid of human CSHY. As long as it does not inhibit or inactivate the activity of hydrolyzing the N-acetyl-D-galactosaminide bond of acetyl-D-galactosamine, it may contain analgesics, anti-inflammatory agents and other pharmacologically active ingredients.

  The pharmaceutical composition of the present invention has a spinal cord injury, for example, spinal cord injury caused by intervertebral disc herniation, particularly epidural migrating disc herniation and transligament prolapse disc herniation, and the nucleus pulposus is removed from the spinal epidural by protrusion or release. It can be used to treat existing types of disc herniation. The therapeutic effect of CSaseABC on spinal cord injury due to hernia has already been confirmed (Ikegami et al., “Clinical Orthopedic Surgery”, 2007, Vol. 42, No. 2, pp. 124-128) and has the activity of degrading chondroitin Like CaseABC, human CSHY also has a therapeutic effect on spinal cord injury, particularly spinal cord injury due to herniated disc.

  In addition, known chondroitinases derived from bacteria such as CSaseABC, which have been studied for use as pharmaceuticals, generate oligosaccharides having uronic acid having an unsaturated bond at the non-reducing end by elimination reaction. Let Unsaturated uronic acid is a sugar that does not exist in mammals and is antigenic to mammals. Therefore, the use of known chondroitinase derived from bacteria has a problem in that it is produced in the human body administered with a substance showing antigenicity to humans. On the other hand, the human CSHY according to the present invention generates an oligosaccharide having glucuronic acid having no unsaturated bond at the non-reducing end by a hydrolysis reaction. Glucuronic acid has a very low antigenicity to mammals. Therefore, a glycosaminoglycan degradation accelerator containing human CSHY or a pharmaceutical composition containing this glycosaminoglycan degradation accelerator and a pharmaceutically inactive ingredient is given to humans. Even if it administers, the problem pointed out by administration of the known chondroitinase derived from bacteria does not occur.

  The present invention relates to a protein comprising the amino acid sequence shown in SEQ ID NO: 1, and an amino acid sequence in which one or several tens of amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 1. A protein having an activity of hydrolyzing an N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine bound to D-glucuronic acid, positions 1-33 and / or 463 in SEQ ID NO: 1 A protein consisting of an amino acid sequence from which the 481st amino acid residue has been deleted, and the amino acid residues 1 to 33 and / or 463 to 481 in SEQ ID NO: 1 have been deleted; Consisting of a substituted, deleted or added amino acid sequence and D-glucuron A step of reacting a glycosaminoglycan with a protein selected from the group consisting of proteins having an activity of hydrolyzing an N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine bound to an acid, and glucuronic acid There is also provided a method for producing an oligosaccharide having glucuronic acid as a non-reducing end from glycosaminoglycan, which comprises a step of recovering an oligosaccharide having a non-reducing end as a non-reducing end.

  The step of reacting the protein with glycosaminoglycan is performed by mixing an appropriate amount of substrate and enzyme protein under the conditions such as pH and temperature at which the glycosaminoglycan degrading activity of human CSHY described above is exerted. Incubating step. Human CSHY is preferably a recombinantly produced protein, and particularly preferably human CSHY immobilized on an appropriate resin. If such solid-phased human CSHY is used, functional glycosaminoglycan can be produced continuously and in large quantities by continuously reacting the solid-phased enzyme protein and glycosaminoglycan. Can do.

  The process of recovering oligosaccharides having glucuronic acid as a non-reducing end is obtained by reacting oligosaccharides having the product glucuronic acid as a non-reducing end from the reaction solution after reacting protein and glycosaminoglycan. Recovering in the form of a liquid, recovering in a partially purified form, or recovering in a substantially fully purified form. In the continuous reaction between the immobilized enzyme protein and glycosaminoglycan as described above, the oligosaccharide that is already a product in the form of a reaction solution separated from the solid support is in a sufficiently purified state. ing. Oligosaccharides are highly purified by various fractionation methods commonly used for purification of oligosaccharides commonly used for gel filtration chromatography, ion (anion, cation) exchange chromatography and other oligosaccharides, or The oligosaccharide may be further finely fractionated for each type of sugar or the number of sugar chains.

  Oligosaccharides that have glucuronic acid as a non-reducing end are expected to exhibit extremely low antigenicity to humans and exhibit anti-inflammatory action, inhibition of cancer metastasis, and other useful functions as glycosaminoglycans, The present invention is useful as a method for producing such a functional oligosaccharide.

  In addition, since human CSHY in the present invention exhibits a high degradation activity on chondroitin sulfate and a mixed polysaccharide containing chondroitin sulfate, particularly on chondroitin sulfate, the glycosaminoglycan degradation accelerator of the present invention and a pharmaceutical comprising the same A preferred embodiment of is a chondroitin degradation promoter, a chondroitin sulfate degradation promoter or a pharmaceutical containing them, and a particularly preferred embodiment is a chondroitin degradation promoter or a pharmaceutical containing the same. Similarly, a preferred embodiment of a method for producing an oligosaccharide from glycosaminoglycan is a method for producing an oligosaccharide from chondroitin sulfate or a hybrid polysaccharide containing chondroitin sulfate, and a particularly preferred embodiment is to produce an oligosaccharide from chondroitin sulfate. It is a method to do.

  Hereinafter, the glycosaminoglycan degradation accelerator according to the present invention will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Example 1> Production of recombinant human CSHY (1) Cloning Based on the nucleotide sequence information of the human HYAL4 gene, the following two sets of primer DNAs are prepared, and nested PCR (nested PCR) using a human placenta cDNA library as a template. Went.

Primer F1 (SEQ ID NO: 3)
5'-GGTTTGGAGCCATGCTGGACATCC-3 '
Primer R1 (SEQ ID NO: 4)
5'-TTTCCTAGCCAGACTGGAGGC-3 '
Primer F2 (SEQ ID NO: 5)
5'-GCACCAAGGTGAACTAAGGACCCA-3 '
Primer R2 (SEQ ID NO: 6)
5′-CATCCCTTCTTTAAATGACTAGGC-3 ′

PCR consists of [94 ° C for 30 seconds, 55 ° C for 45 seconds, 68 ° C for 2 minutes] using KOD-Plus DNA polymerase (Toyobo) in the presence of 5% (v / v) dimethyl sulfoxide. The cycle was performed 30 times. The amplified DNA fragment (about 1.4 kbp) was recovered, ends adding adenosine ^{monophosphate,} it was inserted into the pGEM (R) -T Easy Vector (Promega Corporation). The base sequence of the inserted portion was determined, and it was confirmed that a recombinant vector retaining the DNA shown in SEQ ID NO: 2 (total 1446 bp DNA including the ORF encoding the amino acid sequence of SEQ ID NO: 1) was obtained.

(2) Construction of expression vector PCR was performed using the following set of primers using the recombinant vector obtained in (1) as a template. PCR reaction conditions are the same as in (1).

Primer F3 (SEQ ID NO: 7)
5′-GCGATATCGTGTCTAAAAACCCTCC-3 ′
Primer R3 (SEQ ID NO: 8)
5′-GCGGATCCCTCAAGGAGAAGGGGAAA-3 ′

  The amplified DNA fragment (about 1400 bp) was digested with EcoRV and BamHI and subcloned into the EcoRV site and BamHI site of the expression vector p3XFLAG-CMV-8 (Sigma). By this operation, a fusion sequence in which a leader sequence of preprotrypsin and a 3XFLAG tag are added to the N-terminal side of solubilized human CSHY from which 33 amino acid residues from the N-terminal and 19 amino acid residues from the C-terminal of SEQ ID NO: 1 have been deleted An expression vector retaining a DNA encoding the protein was obtained. The entire amino acid sequence of this fusion protein is as shown in SEQ ID NO: 9.

(3) Expression using recombinant cells Using FuGENE ^(R) 6 (Roche diagnostics), the expression vector (6.7 μg) obtained in (2) was introduced into COS7 cells and cultured at 37 ° C. for 3 days. , and the medium was recovered and the 1 mL, and incubated overnight at ^{ANTI-FLAG (R) M2 affinity} gel and 4 ° C. in 10 [mu] L. Thereafter, the resin was washed with 25 mM Tris-buffered saline (TBS-T) containing 0.1% Tween 20 to obtain a resin adsorbed with the protein contained in the medium.

(4) Confirmation of molecular weight of protein contained in medium The protein adsorbed on the resin obtained in this Example (3) was subjected to Western method and silver staining method according to a conventional method to confirm the molecular weight. The result is shown in FIG.

  As shown in FIG. 1, when the Western method was performed under the silver staining method and the reducing conditions, a clear band of about 80 kDa (indicated by an asterisk in the figure) was confirmed, while in the control, the band of the same molecular weight was It was not confirmed. In addition, when the Western method was performed under non-reducing conditions, a clear band of about 70 kDa (indicated by an asterisk in the figure) was confirmed, whereas a band of the same molecular weight was not confirmed in the control.

  From the above results, it was confirmed that these about 80 kDa band and about 70 kDa band were derived from human CSHY. In addition, since the predicted molecular weight of the prepared fusion protein was 53 kDa, it was suggested that the human CSHY protein contained in the culture solution was subjected to modifications such as glycosylation after translation.

Example 2 Measurement of Chondroitin Sulfate Degradation Promoting Activity ANTI-FLAG ^(R) M2 affinity gel bound with human CSHY prepared in Example 1 (3) was added to TBS-T and 50 mM phosphoric acid containing 150 mM NaCl. After washing with a buffer solution (pH 6.0), chondroitin sulfate A (CS-A) derived from whale cartilage labeled with FITC, chondroitin sulfate C (CS-C) derived from shark cartilage, chondroitin sulfate D (CS) derived from shark fin cartilage -D) and squid cartilage-derived chondroitin sulfate E (CS-E) were resuspended in the same buffer solution containing 10 μg and reacted at 37 ° C. for 12 hours. After removing the resin by filtration using Ultrafree-MC (Millipore), the filtrate was applied to Superdex peptide column (Amersham Biosciences) equilibrated with 0.2 M NH ₄ HCO ₃ for gel filtration. Detection was performed by detecting fluorescence having an excitation wavelength of 490 nm and an emission wavelength of 520 nm. For the activity, the total peak area from RT 19 minutes to 51 minutes is defined as the total fluorescence intensity of chondroitin sulfate labeled with FITC, and the abundance ratio of low molecular weight fragments (RT 34 minutes to 51 minutes) with respect to the total fluorescence intensity is obtained from the peak area. Expressed in The gel filtration elution patterns of various chondroitin sulfates labeled with FITC before the enzyme reaction are shown in panels A, C, E and G of FIG. 2, and the gel filtration elution patterns of the filtrate after the enzyme reaction are shown in panels B and D of FIG. , F and H, respectively.

  As shown in FIG. 2, the peak of chondroitin sulfate D disappeared due to the enzyme reaction, and the peak of oligosaccharide was observed.

Example 3 Analysis of Enzyme Properties (1) Confirmation of Substrate Specificity 10 μL of ANTI-FLAG ^(R) M2 affinity gel bound with human CSHY prepared in Example 1 (3) was mixed with chondroitin, CS-A, CS -C, CS-D, CS-E, chondroitin sulfate B derived from pig skin (CS-B), heparan sulfate derived from bovine kidney, hyaluronic acid (HA), dermatan sulfate derived from pig skin (DS), derived from Ascidia nigra Of dermatan sulfate (2S, 6S-DS) and chondroitin sulfate derived from Ascidia nigra were reacted at 37 ° C. for 12 hours. Each degradation product was fluorescently labeled with 2-aminobenzamide (2AB), applied to Superdex peptide column (GE Healthcare) equilibrated with PBS, and subjected to gel filtration. The results for chondroitin sulfate from CS-D, CS-C, CS-A and Ascidia nigra are shown in panels A, B, C and D of FIG. 3, respectively.

  As shown in panels A, B and C of FIG. 3, it was confirmed from the production amount of the degradation product that human CSHY is substrate-specific in the order of CS-D> CS-C> CS-A. In addition, as shown in Panel D, chondroitin sulfate derived from Ascidia nigra {the structure of a typical constituent disaccharide unit has little effect on iduronic acid (2S) -N-acetyl-D-galactosamine (6S)}. It was. In addition, it was confirmed that human CSHY has a very weak effect on CS-E, and has almost no effect on DS, 2S, 6S-DS, chondroitin, CS-B, heparan sulfate and hyaluronic acid. From the above, it was confirmed that human CSHY is substrate-specific in the order of CS-D> CS-C> CS-A> CS-E.

(2) Optimum temperature 10 μL of ANTI-FLAG ^(R) M2 affinity gel and 10 μg of CS-D to which human CSHY prepared in Example 1 (3) was bound at 20 ° C., 25 ° C., 30 ° C. and 37 ° C. The optimal temperature for human CSHY activity was examined by reacting each for 12 hours. As a result, assuming that the activity measured at 37 ° C. was 1, the activities at 20 ° C., 25 ° C., and 30 ° C. were 0.31, 0.56, and 0.82, respectively (not shown). According to this example, the subsequent experiments were performed at 37 ° C.

(3) Optimum pH
The buffer used in the reaction of Example 2 was adjusted to pH 4.5 to 7.5 with 50 mM phosphate buffer and reacted at 28 ° C. for 9 hours to examine the optimum pH of human CSHY. The result is shown in FIG.

  As shown in FIG. 4, it was confirmed that the optimum pH of human CSHY was pH 5.0.

(4) Kinetic analysis Based on the Lineweaver-Burk plot prepared from the initial reaction rate and the concentration change of the substrate (disaccharide), CS-D, CS-A, CS-C and CS of chondroitin sulfate isoforms of human CSHY The maximum reaction rate Vmax when each of -E was used as a substrate and the apparent Michaelis constant Km were determined. The results are shown in FIG. 5 and the following Table 1.

  As shown in FIG. 5 and Table 1, the Km values when CS-D (panel C in FIG. 5) is used as a substrate are CS-A (panel A in FIG. 5), CS-C (panel B in FIG. 5). ) And CS-E (panel D in FIG. 5) were 1/2, 1/3, and 1/20, respectively, as compared to the Km values when using as a substrate. That is, it was confirmed that the reaction rate of each chondroitin sulfate isoform that is sensitive to human CSHY was different and that CS-D was the most preferred substrate among the chondroitin sulfates used as the substrate. This result was consistent with the results obtained by quantifying the reducing end produced by reacting human CSHY with various glycosaminoglycans under the same conditions (panels A to C in FIG. 3).

<Example 4> Analysis of sugar chain sequence recognized by human CSHY (1) Identification of reducing end of reaction product In order to examine the activity of human CSHY in more detail, CS-D is digested with human CSHY, and the resulting degradation product After the product was fluorescently labeled with 2AB, gel filtration HPLC was performed with Superdex peptide column (GE Healthcare). As a result, three main peak extracts D-1, D-2, and D-3, each identified as tetrasaccharide, hexasaccharide, and octasaccharide, were obtained (Panel A in FIG. 3).

  As a result of analyzing this extract D-1 by anion exchange HPLC, a main peak identified by 2AB-tetrasaccharide trisulfate was obtained (not shown). Since the main component contained in this peak cannot be obtained with CSase AC-II, the carbon at the 2-position is sulfated (2-0-sulfate in most of the internal GlcA residues contained in the extract D-1. ) Was strongly suggested.

  Subsequently, in order to determine the main sugar sequences contained in the extracts D-1, D-2 and D-3, first, these were each digested with CSase AC-II, and the resulting degradation products were each 2AB. After fluorescent labeling with, it was analyzed by anion exchange HPLC.

  Moreover, the human CSHY degradation product of CS-D was monitored by UV absorbance at 215 nm without being fluorescently labeled with 2AB, and gel filtration HPLC was performed, and then the peak of tetrasaccharide, which is the lowest molecule, was recovered. The recovered tetrasaccharide was decomposed with CSaseABC, fluorescently labeled with 2AB, and analyzed by anion exchange HPLC.

  Furthermore, extracts D-2 and D-3 were each digested with CSaseABC, and the resulting degradation products were fluorescently labeled with 2AB, and then analyzed by anion exchange HPLC. The above results are shown in FIG.

  In FIG. 6, panels A to C show the elution pattern of products obtained by digesting fractions D-1, D-2 and D-3 with CSase AC-II, and panel D shows human CSHY not fluorescently labeled with 2AB. 4 shows the elution pattern of a product obtained by isolating the tetrasaccharide, which is the lowest molecule among the degradation products, and further digesting with CSaseABC and then fluorescently labeling the resulting degradation product with 2AB. Panels E and F show the elution patterns of samples fluorescently labeled with 2AB after fractions D-2 and D-3 were digested with CSaseABC, respectively. Each arrow in Panels C and F shows a peak a: ΔHexA (2-O-sulfate) -GalNAc (6-O-sulfate) -GlcA-GalNAc (6-O-sulfate), peak b: ΔHexA (2-O -Sulfate) -GalNAc (6-O-sulfate) -GlcA-GalNAc (4-O-sulfate), peak c: ΔHexA-GalNAc (4-O-sulfate) -GlcA (2-O-sulfate) -GalNAc (6 -O-sulfate), peak d: [Delta] HexA-GalNAc (6-O-sulfate) -GlcA (2-O-sulfate) -GalNAc (6-O-sulfate)), peak 1: [Delta] HexA-GalNAc, peak 2: [Delta] HexA -GalNAc (6-O-sulfa e), peak 3: ΔHexA-GalNAc (4-O-sulfate), peak 4: ΔHexA (2-O-sulfate) -GalNAc (6-O-sulfate), peak 5: ΔHexA (2-O-sulfate) − GalNAc (4-O-sulfate), peak 6: ΔHexA-GalNAc (4,6-O-sulfate), peak 7: ΔHexA (2-O-sulfate) -GalNAc (4,6-O-sulfate), peak 8 : GlcA-GalNAc (6-O-sulfate) and peak 9: The elution position of GlcA-GalNAc (4-O-sulfate) is shown.

  As shown in panel A of FIG. 6 and Table 2 (D-1), extract D-1 is identified by GlcA-GalNAc (4S) -2AB and ΔHexA (2S) -GalNAc (6S) -2AB. Two main peaks were obtained. That is, it was confirmed that the main sugar sequence contained in the tetrasaccharide fraction of the CS-D human CSHY degradation product was GlcA-GalNAc (4S) -GlcA (2S) -GalNAc (6S). For extract D-2, a peak indicating 2AB-tetrasaccharide trisulfate was obtained as shown in panel B of FIG. 6, and extract D-3 was also shown in panel C of FIG. Similar results were obtained. Each of these degradation products was analyzed by anion exchange HPLC together with a 2AB-unsaturated tetrasaccharide standard having a known structure, and as a result, each was eluted with ΔHexA-GalNAc (4S) -GlcA (2S) -GalNAc (6S) -2AB. (Table 2 (D-2) and (D-3)). From this, it was confirmed that human CSHY recognizes the sequence of the tetrasaccharide GlcA-GalNAc (4S) -GlcA (2S) -GalNAc (6S) as a structure on the non-reducing terminal side in the cleavage site of CS-D. . The above results are summarized and shown in Table 3 below.

  On the other hand, since CS-A and CS-C are also digested to some extent by human CSHY (FIGS. 2 and 3), the reducing terminal structure of oligosaccharides produced by digesting CS-A and CS-C to human CSHY Was analyzed for the specificity of human CSHY. Specifically, CS-A and CS-C were digested with human CSHY, and the resulting degradation product was fluorescently labeled with 2AB, further digested with CSase AC-II, and then analyzed by anion exchange HPLC. The result is shown in FIG.

  In FIG. 7, arrows 1 to 7 indicate 1: ΔHexA-GalNAc, 2: ΔHexA-GalNAc (6-O-sulfate), 3: ΔHexA-GalNAc (4-O-sulfate), 4: ΔHexA (2-O -Sulfate) -GalNAc (6-O-sulfate), 5: [Delta] HexA (2-O-sulfate) -GalNAc (4-O-sulfate), 6: [Delta] HexA-GalNAc (4,6-O-sulfate) and 7: Each elution position of 2AB derivative of ΔHexA (2-O-sulfate) -GalNAc (4,6-O-disulphate) is shown. The white arrow indicates the elution position of ΔHexA-GalNAc (4-O-sulfate) -GlcA (2-O-sulfate) -GalNAc (6-O-sulfate), which is a standard tetrasaccharide fluorescently labeled with 2AB. Show.

  As shown in panel A of FIG. 7, the only reaction product derived from the reducing end contained in the human CSHY degradation product of CS-A was detected as ΔHexA-GalNAc (6S) -2AB. That is, it was confirmed that the main reducing terminal disaccharide in the human CSHY degradation product of CS-A was GlcA-GalNAc (6S) (Table 3). Since the main disaccharide constituting CS-A is not GlcA-GalNAc (6S) (24%) but GlcA-GalNAc (4S) (76%), the sulfation of the 6-position carbon of the GalNAc residue is prevented. It was confirmed that it is essential for recognition of the cleavage site by human CSHY.

  Further, as shown in panel B of FIG. 7, for CS-C, ΔHexA-GalNAc (6S) -2AB and ΔHexA-GalNAc (4S) -GlcA (2S) -GalNAc (6S) -2AB are the main oligosaccharides. The molar ratio was 1.2: 1.0. That is, it was confirmed that the main reducing end disaccharide sequences in the human CSHY degradation product of CS-C were GlcA-GalNAc (6S) and GlcA (2S) -GalNAc (6S) (Table 3).

  The molar ratio of these GlcA-GalNAc (6S) and GlcA (2S) -GalNAc (6S) in CS-C is 10.7: 1.0. That is, the ratio of GlcA-GalNAc (6S) in CS-C is considerably larger than that of GlcA (2S) -GalNAc (6S), but these are the main reducing ends contained in the human CSHY degradation product of CS-C. As disaccharides, they are detected at approximately the same ratio. This suggests that human CSHY is more likely to select GlcA (2S) -GalNAc (6S) than GlcA-GalNAc (6S) as the cleavage site for CS-C. That is, it is considered that sulfation of the carbon at position 2 of the GlcA residue adjacent to the non-reducing terminal side of the GalNAc (6S) residue promotes human CSHY activity. Here, the schematic diagram which shows the structure of the site | part cut | disconnected by human CSHY in CS-C is shown in FIG. In FIG. 8, the sulfate group that has been cleaved at the arrowed portion and marked with * indicates that it has a promoting effect on the hydrolytic activity of human CSHY.

(2) Identification of non-reducing end of reaction product In order to investigate the structure of the reducing end side at the cleavage site of CS-D, the non-reducing end disaccharide contained in Extract D-2 and Extract D-3 was examined. Specifically, the hexasaccharide fraction of the extract D-2 was decomposed with CSaseABC and fluorescently labeled with 2AB, and then analyzed by anion exchange HPLC. As a result, GlcA-GalNAc (4S) -2AB, GlcA-GalNAc ( Three peaks of 6S) -2AB and ΔHexA-GalNAc (4S) -GlcA (2S) -GalNAc (6S) -2AB were obtained. These molar ratios were 0.35, 0.40, and 1.0, respectively (Panel E in FIG. 6 and Table 2). That is, the extract D-2 fraction was GlcA-GalNAc (4S) -GlcA-GalNAc (4S) -GlcA (2S) -GalNAc (6S) and GlcA-GalNAc (6S) -GlcA-GalNAc (4S) -GlcA ( It was confirmed that the two components of 2S) -GalNAc (6S) were contained in a molar ratio of 47:53 (Table 3).

  Further, the octasaccharide fraction of the extract D-3 was decomposed with CSaseABC and fluorescently labeled with 2AB, and then analyzed by anion exchange HPLC. As a result, ΔHexA-GalNAc (4S) -GlcA (2S) -GalNAc (6S) derived from two saturated disaccharides, three unsaturated disaccharides, and the reducing end was obtained (panel F in FIG. 6). These ratios are shown in Table 2, and the deduced sequences of main octasaccharides in Extract D-3 are shown in Table 3.

  As shown in Table 2 and Table 3, it can be seen that the saturated disaccharide and the unsaturated disaccharide are derived from the non-reducing end and the inside of the octasaccharide contained in the extract D-3 fraction, respectively.

<Example 5> Identification of amino acids important for the degradation activity of human CSHY For the four amino acids that may be important for the degradation activity of human CSHY, the base sequences encoding the following variants 1 to 4 were determined in a conventional manner. According to the above, it was prepared by a PCR method using QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene).

  Specifically, the following four sets of primer DNAs were prepared, and PCR was performed in the same manner as in Example 1 (2) using the recombinant vector prepared in Example 1 (1) as a template. The PCR reaction solution composition and PCR reaction conditions followed the protocol of QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene).

Primer F1 (SEQ ID NO: 10)
5′-CTGTTATAGATTGGCAAATTGGGCACCAC-3 ′
Primer R1 (SEQ ID NO: 11)
5'-GTGGTCGCCCAAATTGCCACATCTAATAAGAG-3 '
Primer F2 (SEQ ID NO: 12)
5'-GGCCCTTTGGGGTTATGCTTTATATCCT-3 '
Primer R2 (SEQ ID NO: 13)
5'-CAGGATAATAAGAGATAACCCCCAAAGGCC-3 '
Primer F3 (SEQ ID NO: 14)
5′-ATATCCTTCTCATTCTGTCGGAAATCCC-3 ′
Primer R3 (SEQ ID NO: 15)
5'-GGGATTTCCACACATAGATAGAAGGADAT-3 '
Primer F4 (SEQ ID NO: 16)
5'-GGGAGCTACATACGCCGCTGTGACCAG-3 '
Primer R4 (SEQ ID NO: 17)
5′-CTGGTCACAGCGGCTATGTAGCTCCCC-3 ′

Nucleotide sequence variant 1 encoding mutant human CSHY prepared; nucleotide sequence (SEQ ID NO: 18) encoding mutant human CSHY (SEQ ID NO: 19) in which the 147th glutamic acid in the amino acid sequence of SEQ ID NO: 1 was replaced with glutamine
Mutant 2: a nucleotide sequence (SEQ ID NO: 20) encoding a mutant human CSHY (SEQ ID NO: 21) in which the 218th tyrosine is substituted with alanine in the amino acid sequence of SEQ ID NO: 1
Mutant 3; nucleotide sequence (SEQ ID NO: 22) encoding mutant human CSHY (SEQ ID NO: 23) in which the 263th glycine in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine
Mutant 4: a nucleotide sequence (SEQ ID NO: 24) encoding a mutant human CSHY (SEQ ID NO: 25) in which the 368th asparagine is substituted with alanine in the amino acid sequence of SEQ ID NO: 1

  The prepared mutants 1 to 4 were expressed in recombinant cells by the same method as in Example 1 (1) to Example 1 (3), and a resin adsorbing these mutant proteins was prepared. Subsequently, the degradation activity for CS-D was examined by the same method as in Example 3 (1). The result is shown in FIG.

  As shown in FIG. 9, it was revealed that Mutant 1, Mutant 2 and Mutant 4 lost the degradation activity for CS-D. On the other hand, it was revealed that the mutant 3 has a degradation activity equivalent to that of the control wild type human CSHY.

  From the above results, it was confirmed that the 147th glutamic acid, the 218th tyrosine and the 368th asparagine in the amino acid sequence of SEQ ID NO: 1 are important in the degradation activity of human CSHY on CS-D.

Claims (12)

  A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or consisting of an amino acid sequence in which one or several tens of amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and D- A glycosaminoglycan degradation promoter comprising, as an active ingredient, a protein having an activity of hydrolyzing an N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine bound to glucuronic acid.   A protein comprising an amino acid sequence in which amino acid residues 1 to 33 and / or 463 to 481 are deleted in SEQ ID NO: 1, or amino acid residues 1 to 33 and / or 463 to 481 in SEQ ID NO: 1 The glycosaminoglycan degradation promoter according to claim 1, wherein the active ingredient is a protein having an amino acid sequence that is deleted and further substituted, deleted, or added with one or several amino acids.   The glycosaminoglycan degradation accelerator according to claim 1 or 2, wherein the glycosaminoglycan is chondroitin sulfate or a hybrid polysaccharide containing chondroitin sulfate.   The glycosaminoglycan degradation promoter according to claim 3, wherein the chondroitin sulfate is one or more chondroitin sulfates selected from the group consisting of chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D and chondroitin sulfate E.   The glycosaminoglycan degradation promoter according to any one of claims 1 to 4, wherein the protein is in the form of a fusion protein.   A pharmaceutical composition comprising the glycosaminoglycan degradation accelerator according to any one of claims 1 to 5 and a pharmaceutically inactive ingredient.   The pharmaceutical composition according to claim 6 for treating spinal cord injury.   The pharmaceutical composition according to claim 7, wherein the spinal injury is an injury caused by an herniated disc.   The pharmaceutical composition according to claim 8, wherein the disc herniation is an epidural disc herniation or a transligament prolapse disc herniation.   A method for producing an oligosaccharide having glucuronic acid as a non-reducing end from glycosaminoglycan, comprising a protein comprising the amino acid sequence represented by SEQ ID NO: 1, one or several tens of amino acids in the amino acid sequence represented by SEQ ID NO: 1 Activity of hydrolyzing the N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine consisting of an amino acid sequence substituted, deleted, or added and bound to D-glucuronic acid A protein having an amino acid sequence in which amino acid residues 1 to 33 and / or 463 to 481 in SEQ ID NO: 1 are deleted, and 1 to 33 and / or 463 to 481 in SEQ ID NO: 1 Amino acid residues are deleted and one or more amino acids are placed A protein having an activity of hydrolyzing an N-acetyl-D-galactosaminide bond of sulfated N-acetyl-D-galactosamine which is composed of an amino acid sequence which is deleted, deleted or added and which is bound to D-glucuronic acid The method comprising the steps of reacting one or more proteins selected from the group consisting of glycosaminoglycan and recovering an oligosaccharide having glucuronic acid as a non-reducing end.   The method for producing an oligosaccharide according to claim 10, wherein the glycosaminoglycan is chondroitin sulfate or a hybrid polysaccharide containing chondroitin sulfate.   The method for producing an oligosaccharide according to claim 11, wherein the chondroitin sulfate is one or more chondroitin sulfates selected from the group consisting of chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D and chondroitin sulfate E.

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